Substances k01-0509 and process for producing the same

ABSTRACT

A microorganism belonging to the genus  Streptomyces  and being capable of producing substance K01-0509-A1 and/or substance K01-0509-A2 is cultured in a medium. Then, the substance K01-0509-A1 and/or the substance K01-0509-A2 having been thus accumulated in the liquid culture medium are collected from the medium. It is expected that the obtained substances are efficacious as drugs which are selectively effective on pathogenic gram-negative bacteria having the type III secretion system.

TECHNICAL FIELD

The present invention relates to K01-0509 substance which inhibits typeIII secretion mechanism of bacteria and a process for productionthereof. More particularly the present invention pertains to K01-0509substance comprising K01-0509-A1 substance and/or K01-0509-A2 substancewhich are useful as a remedy or preventive for infectious disease.

BACKGROUND ART

The type III secretion mechanism, which is a function for releasingbacterial pathogenic factor to the extracellular field, has beenreported to be highly conserved, for example, in bacteria of genusSalmonella, genus Yersinia, genus Pseudomonas, Shigella,enteropathogenic E. coli (hereinafter sometimes designates as EPEC),enterohemorrhagic E. coli and genus Bordetella (Microbiology andMolecular Biology Reviews, June, 1998, p. 381).

It has been reported that bacteria maintaining the type III secretionmechanism hereinabove released the pathogenic factor into theextracellular field through the secretion mechanism and a part of thereleased pathogenic factor was transferred into the host cell, and thepathogenic factor transferred into the host cell was largely involved inthe pathogenicity of bacteria (Microbiology and Molecular BiologyReviews, June, 1998, p. 382 ff.).

On the other hand, it was demonstrated that EPEC strain defective intype III secretion system (hereinafter sometimes designates as type IIIsecretion protein) lost the pathogenicity in the infectious experimentsusing rabbits (J. Exp. Med. 188(10), 1907-1916, 1998, November 16) andthe infectious experiment using human volunteers (Infection andImmunity, June 2000, p. 3689-3695). From these facts, substancesinhibiting the type III secretion system and the function of thesecretion protein are expected to exhibit effects as a remedy orpreventive remedy for infectious disease having new ideas withoutkilling bacteria but making the pathogenicity disappear.

DISCLOSURE OF THE INVENTION

In such circumstances, problem to be solved by the present invention isto find out new antiinfectious disease agents which allow to disappearthe pathogenicity of pathogenic bacteria.

In order to solve such problems hereinabove described, we have continuedstudies on microbial metabolites, and found that substances havinginhibitory activities against the type III secretion system wereproduced in the cultured medium of a newly isolated strain, designatedas K01-0509, from soil sample collected in Amami-Oshima. Further, wehave found that substances represented by the chemical structure of theformula [I] and [II] hereinbelow as a result of isolation andpurification of the active principle from the cultured mass showinginhibitory activities against the type III secretion system. Sincesubstances having such chemical structures were not known, thesesubstances were designated as K01-0509-A1 substance and K01-0509-A2substance, and were totally designated as K01-0509 substance.

The present invention has been completed by such knowledge.

An aspect of the present invention is to provide K01-0509-A1 substancerepresented by the following formula [I];

An aspect of the present invention is to provide K01-0509-A2 substance(a stereoisomer of A1 substance) represented by the following formula[II];

Further aspect of the present invention is to provide K01-0509Asubstance consisting of K01-0509-A1 substance represented by thefollowing formula [I];

and K01-0509-A2 substance represented by the following formula [II];

Another aspect of the present invention is to provide a process forproduction of K01-0509-A1 substance comprising culturing microorganismbelonging to genus Streptomyces and having ability to produceK01-0509-A1 substance, accumulating K01-0509-A1 substance in a culturedmedium and isolating K01-0509-A1 substance from the cultured mass.

More further aspect of the present invention is to provide a process forproduction of K01-0509-A2 substance comprising culturing microorganismbelonging to genus Streptomyces and having ability to produceK01-0509-A2 substance, accumulating K01-0509-A2 substance in a culturedmedium and isolating K01-0509-A2 substance from the cultured mass.

Further aspect of the present invention is to provide a process forproduction of a composition consisting of K01-0509-A1 substance andK01-0509-A2 substance comprising culturing microorganism belonging togenus Streptomyces and having ability to produce K01-0509-A1 substanceand K01-0509-A2 substance, accumulating K01-0509-A1 substance andK01-0509-A2 substance in a cultured medium and isolating K01-0509-A1substance and K01-0509-A2 substance from the cultured mass.

Further aspect of the present invention is to provide a process forproduction of K01-0509-A1 substance wherein a microorganism belonging togenus Streptomyces and having ability to produce K99-05278-A1 substanceis Streptomyces sp. K01-0509 FERM BP-08504.

Further aspect of the present invention is to provide a process forproduction of K01-0509-A2 substance wherein a microorganism belonging togenus Streptomyces and having ability to produce K99-05278-A2 substanceis Streptomyces sp. K01-0509 FERM BP-08504.

Further aspect of the present invention is to provide a process forproduction of a composition consisting of K01-0509-A1 substance andK01-0509-A2 substance wherein a microorganism belonging to genusStreptomyces and having ability to produce K99-05278-A1 substance andK01-0509-A2 substance is Streptomyces sp. K01-0509 FERM BP-08504.

Further aspect of the present invention is to provide a microorganismwhich is Streptomyces sp. K01-0509 FERM BP-08504.

The microorganism having ability to produce K01-0509-A1 substance andK01-0509-A2 substance or a composition thereof represented by theformula [I] and [II] hereinbefore (hereinafter designates as “K01-0509substance producing microorganism”) belongs to genus Streptomyces, and,for example, Streptomyces sp. K01-0509, which was newly isolated by thepresent inventors, is the most preferable strain used in the presentinvention.

Taxonomical properties of Streptomyces sp. K01-0509 of the presentinvention are as follows.

(I) Morphological Properties

Vegetative mycelia grow well on various agar media and no fragmentationis observed. Aerial mycelia are abundantly grown on yeast-malt extractagar medium and glycerol-asparagine agar medium, and exhibit white tograyish color. On microscopic observation, chains of more than 20 sporesare observed on the aerial mycelia, and the morphological form is linearchains and size of spore is about 0.6-0.8×1.0-1.8 μm with cylindricalform. Surface of the spore is smooth. Sclerotia, sporangia and zoosporesare not observed.

(II) Culture Properties on Various Media

Culture properties of the producing strain of the present inventiondetermined by the method of E. B Shirling and D. Gottlieb (InternationalJournal of Systematic Bacteriology, 16: 313, 1966) are shown in thefollowing. Color tone was determined referring to Color Harmony Manual,4th Ed. (Container Corporation of America, Chicago, 1958) as a standardcolor, and color name as well as attached code number in theparenthesis. Unless otherwise noted, results are observation of culturesat 27° C. for 2 weeks on various media. Culture propertiesSucrose-nitrate agar medium Growth good growth, light amber (3ic)Reverse side light amber (3ic) Aerial mycelium moderate epiphytic, white(a)-gray (h) Soluble pigment none Glucose-asparagine agar medium Growthgood growth, pearl pink (3ca) Reverse side pearl pink (3ca) Aerialmycelium none Soluble pigment none Glycerol-asparagine agar medium (ISP)Growth good growth, light amber (3ic) Reverse side light amber (3ic)Aerial mycelium abundantly epiphytic, white (a)-gray (f) Soluble pigmentnone Starch-inorganic salt agar medium (ISP) Growth good growth, lightamber (3ic) Reverse side light tan (3gc)-light amber (3ic) Aerialmycelium moderate epiphytic, white (a)-gray (h) Soluble pigment noneTyrosine agar medium (ISP) Growth good growth, bamboo (2gc) Reverse sidebamboo (2gc) Aerial mycelium moderately epiphytic, white (a) Solublepigment none Oatmeal agar medium (ISP) Growth good growth, light amber(3ic) Reverse side light tan (3gc)-orange rust (4pe) Aerialmyceliummoderately epiphytic, white (a)-dark cobalt gray (2ih) Soluble pigmentnone Yeast-malt extract agar medium (ISP) Growth good growth, amber(3lc) Reverse side light amber (3ic)-dark luggage tan (4pg) Aerialmycelium abundantly epiphytic, white (a)-gray (g) Soluble pigment noneNutrient agar medium Growth good growth, pearl pink (3ca) Reverse sidepearl pink (3ca) Aerial mycelium none Soluble pigment nonePeptone-yeast-iron agar medium (ISP) Growth good growth, bamboo (2fb)Reverse side bamboo (2fb) Aerial mycelium poorly epiphytic, light ivory(2ca) Soluble pigment none Glucose-nitrate agar medium Growth moderategrowth, bright (3ia) Reverse side orange (4la) Aerial mycelium noneSoluble pigment none Glycerol-calcium malate agar medium Growth goodgrowth, bamboo (2fb) Reverse side pearl pink (3ca) Aerial myceliumpoorly epiphytic, white (a) Soluble pigment none Glucose-peptone agarmedium Growth good growth, light ivory (2ca) Reverse side pearl pink(3ca) Aerial mycelium none Soluble pigment none

(III) Physiological Properties (1) Formation of melanin pigment (a)Tyrosine agar negative (b) Peptone-yeast-iron agar negative medium (c)Tryptone-yeast liquid positive (d) Simple gelatin medium positive(21-23° C.) false (2) Nitrate reduction negative (3) Liquefaction ofgelatin negative (21-23° C.) (simple gelatin medium) (4) Starchhydrolysis positive (5) Coagulation of defatted milk positive (37° C.)(6) Peptonization of defatted milk positive (37° C.) (7) Growthtemperature 10-38° C. (8) Utilization of carbon sources (Pridham-Gottlieb agar medium) Utilize: D-glucose, L-arabinose Not utilize:D-xylose, D-mannitol, L-rhamnose, D-fructose, myo-inositol, raffinose,melibiose, sucrose (9) Decomposition of cellulose negative(IV) Composition of Cell Wall

2,6-diaminopimelic acid of cell wall is LL type. Main menaquinone isMK-9(H₆) and MK-9 (H₈).

(V) Conclusion

Taxonomical properties of the strain of the present invention aresummarized as follows. 2,6-diaminopimelic acid in the cell wall is LLtype and main menaquinone is MK-9 (H₆) and MK-9 (H₈). Morphology of thespore chain is linear chain, forming with long spore chains and smoothspore surface. Various properties on the culture are exhibiting browncolor tone vegetative mycelia and white to grayish aerial mycelia.Production of melanin pigment is observed in Tryptone-yeast liquid.

According to results hereinabove, the present strain was identified asthe strain belonging to genus Streptomyces based on descriptions in“Bergey's Manual of Systematic Bacteriology, Vol. 4, 1989”.

The strain was deposited, based on Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure, as Streptomyces sp. K01-0509 in International Patent OrganismDepository National Institute of Advanced Industrial Science andTechnology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, 305-8566 Japan on October 6, 2003 as permanent depositorynumber FERM BP-08504.

The strain of Streptomyces sp. K01-0509 can be mentioned as a preferableexample of K05-0509 substance producing strain used in the presentinvention. However, since the morphological properties of microorganismsare generally very easily mutated and are not constant. Natural mutationor artificial mutation generally performed by ultraviolet irradiation orchemical mutagens such as N-methyl-N′-nitro-N-nitrosoguanidine and ethylmethansulfonate, are well known. The strain belonging to genusStreptomyces and having ability to produce K01-0509 substancerepresented by the formula [I] and [II] hereinbefore, including theartificial mutants as well as natural mutants or composition thereof,can be used in the present invention.

In a production of K01-0509 substance of the present invention, atfirst, K01-0509 substance producing strain belonging to genusStreptomyces is cultured in a preferable medium. Nutrient sourcespreferable for production of K01-0509 substance of the present inventionare assimilable carbon sources for microorganism, digestible nitrogensources and, if necessary, inorganic salts. Examples of assimilablecarbon sources are sugars such as glucose, fructose, maltose, lactose,galactose, dextrin and starch, and plant oil such as soybean oil, etc.are used independently or in combination.

Examples of nitrogen sources are peptone, yeast extract, meat extract,soybean powder, cotton seed powder, corn steep liquor, malt extract,casein, amino acids, urea, ammonium salts and nitrates are usedindependently or in combination. If necessary, salts such as phosphate,magnesium, calcium, sodium, potassium, heavy metallic salts such asiron, manganese, copper, cobalt or zinc, vitamins and substancessuitable for production of K01-0509 substance are added.

In the liquid culture, if foaming occurs, antifoam agents such as liquidparaffin, animal oil, vegetable oil, silicone oil and surface activeagent can preferably be added. The above culture can be performed byliquid or solid culture condition, if the above nutrient sources arecontained, and in general, the culture can preferably be performed usingliquid culture medium, and in case of small production, the cultureusing flask is preferable.

In the large scale production using the large tank, in order to preventdelay of growth of microorganism in the production process, theproduction strain is inoculated and cultured initially in relativelysmall amount of culture medium, subsequently the cultured mass istransferred into the large tank and cultivation is preferably continued.In this case, compositions of the medium used in the pre-culture and themedium used in the production culture can be identical or different ifnecessary.

In the culture under aeration spinning condition, conventional means,for example, agitation using propeller and other mechanical stirring,rotation or shaking in fermenter, treating with pumping and blowing aircan be applied. Air for aeration should be sterilized. Culturingtemperature can be applied within ranges in the production of K01-0509substance by K01-0509 substance producing strain, and the cultivation isperformed usually at 20-30° C., preferably at 27° C. Culturing pH isusually pH 5-8, preferably about pH 7. Culturing time depends onculturing condition and is usually for 3 days. The thus obtainedaccumulated K01-0509 substance in the cultured mass exists generally incultured supernatant. Isolation of K01-0509 substance from the culturedmedium can be performed by methods used for isolation of metabolitesfrom microbial cultured mass independently, repeatedly or in combinationwith any orders of the means.

Isolation and collection of K01-0509 substance can be performed bycollecting from the cultured supernatant. The cultured supernatant istreated by the known method used for collection of water solublesubstance, for example, chromatography such as adsorptionchromatography, gel filtration chromatography and high performanceliquid chromatography, in combination or repetition thereof to isolateK01-0509 substance.

Physicochemical properties of K01-0509 substance of the presentinvention are explained hereinbelow.

-   1. K01-0509-A1 substance    -   (1) Nature: white powder    -   (2) Molecular formula: C₂₃H₄₀N₈O₈        -   HRFAB-MS(m/z)[M+H]⁺        -   Calculated 557.3047, Found 557.3052    -   (3) Molecular weight: 556        -   FAB-MS(m/z) [M+H]⁺ 557, [M+Na]⁺ 579    -   (4) Ultraviolet absorption spectrum (in water): as shown in FIG.        1, terminal absorption    -   (5) Infrared absorption spectrum (KBr Tablet): as shown in FIG.        2, specific maximum absorption λmax at 1564, 1682 cm⁻¹.    -   (6) Specific rotation: [α]_(D) ²=−5.00° (c=0.1, methanol)    -   (7) Solubility in solvent: soluble in water, dimethyl sulfoxide        (DMSO) and methanol, insoluble in acetonitrile, ethyl acetate,        chloroform and acetone.    -   (8) Grouping for acidic, neutral and basic: Basic substance.    -   (9) Amino acid analysis: L-alanine and L-valine (1:1).    -   (10) ¹H nuclear magnetic resonance spectrum (in deuterium oxide)        measured by using Varian NMR 400 MHz (FIG. 3). Chemical shifts        of hydrogen (ppm) are: 0.92(3H), 0.92(3H), 1.39(3H), 1.44,        1.55(3H), 1.57, 1.71, 1.76, 1.77, 1.93, 2.11, 3.48, 3.61, 3.79,        3.97, 4.07, 4.17, 4.23, 4.27, 4.43 and 4.44.    -   (11) ¹³C-nuclear magnetic resonance spectrum (in deuterium        oxide) measured by using Varian NMR 100 MHz (FIG. 4). Chemical        shifts of carbon (ppm) are: 18.2, 19.1, 20.1, 21.1, 29.6, 30.3,        32.5, 38.8, 52.2, 53.1, 53.3, 53.8, 54.0, 56.0, 62.4, 74.1,        76.7, 158.9, 158.9, 168.4, 172.1, 176.6 and 179.2.

As shown in above, as a result of detailed examination of variousphysico-chemical properties and spectral data of K01-0509-A1 substance,K01-0509-A1 substance was determined to have the chemical structure asshown in the formula [I].

-   2. K01-0509-A2 substance    -   (1) Nature: white powder    -   (2) Molecular formula: C₂₃H₄₀N₈O₈        -   HRFAB-MS(m/z)[M+H]⁺        -   Calculated 557.3047, Found 557.3023    -   (3) Molecular weight: 556        -   FAB-MS(m/z) [M+H]⁺ 557, [M+Na]⁺ 579    -   (4) Ultraviolet absorption spectrum (in water): as shown in FIG.        5, terminal absorption    -   (5) Infrared absorption spectrum (KBr Tablet): as shown in FIG.        6, specific maximum absorption λmax at 1564, 1682 cm⁻¹.    -   (6) Specific rotation: [α]_(D) ²⁶=−7.4° (c=0.1, methanol)    -   (7) Solubility in solvent: soluble in water, dimethyl sulfoxide        (DMSO) and methanol, :insoluble in acetonitrile, ethyl acetate,        chloroform and acetone.    -   (8) Grouping for acidic, neutral and basic: Basic substance.    -   (9) Amino acid analysis: L-alanine and L-valine (1:1).    -   (10) ¹H nuclear magnetic resonance spectrum (in deuterium oxide)        measured by using Varian NMR 400 MHz (FIG. 7). Chemical shifts        (ppm) are: 0.92(3H), 0.92(3H), 1.39(3H), 1.44, 1.53(3H), 1.57,        1.71, 1.76, 1.77, 1.93, 2.12, 3.48, 3.61, 3.79, 3.99, 4.07,        4.09, 4.23, 4.27, 4.44 and 4.50.    -   (11) ¹³C-nuclear magnetic resonance spectrum (in deuterium        oxide) measured by using Varian NMR 100 MHz (FIG. 8). Chemical        shifts (ppm) are: 17.0, 19.2, 20.1, 21.1, 30.1, 30.4, 32.6,        38.8, 52.2, 53.2, 53.8, 54.0, 54.9, 59.8, 62.4, 74.2, 76.8,        158.9, 159.9, 168.3, 172.1, 176.6 and 179.0.

As shown in above, as a result of detailed examination of variousphysico-chemical properties and spectral data of K01-0509-A2 substance,K01-0509-A2 substance was determined to have the chemical structure asshown in the formula [II].

Biological properties of K01-0509 substance of the present invention areexplained in detail hereinbelow. Inhibitory activity against the typeIII secretion system and antibacterial activity were assayed by thefollowing methods.

(1) Assay of Enteropathogenic E. coli Type III Secretion SystemDependent Hemolytic Inhibitory Activity

Test was performed according to method for detecting substancesinhibiting the bacterial type III secretion system and function ofsecretory proteins thereof which was established by Omura et al. (WO02/057760A1 and corresponding U.S. Pat. No. 6,586,200). One loopfulenteropathogenic E. coli cesT defective strain (WO 02/057760A1) wasinoculated in LB liquid medium (EB media 2.5%, Funakoshi Co. Ltd.,Japan) (5 ml) and cultured at 37° C. for 12 hours without shaking.Bacterial culture (1%) was inoculated to M9 medium containing casaminoacid (sodium dihydrogen phosphate 0.68% (Kanto Chemical Inc., Japan),potassium dihydrogen phosphate 0.3% (Wako Pure Chemical Industries Ltd.,Japan), sodium chloride 0.05% (Kanto Chemical Inc., Japan), ammoniumchloride 0.1% (Wako Pure Chemical Industries Ltd., Japan), glucose 0.4%(Wako Pure Chemical Industries Ltd., Japan), casamino acid 0.1% (SankoJunyaku Co., Ltd., Japan) and magnesium sulfate 0.012% (Kanto ChemicalInc., Japan)) and cultured at 37° C. for further 4 hours withoutshaking. Cultured liquid was centrifuged at 3500 rpm for 15 minutes tocollect bacterial cell precipitation. The bacterial pellets weresuspended in the fresh M9 medium (5 ml) to prepare the test bacterialliquid.

Erythrocytes was washed three times by centrifugation of theerythrocytes suspension, which was prepared by adding physiologicalsaline (40 ml) to the sheep red blood cells (8 ml) (obtainable fromNippon Biological Materials Center, Japan), at 4° C. at 2500 rpm for 5minutes. Weight of the erythrocyte pellets was measured. M9 mediumcontaining casamino acid (2 ml) was added to the erythrocytes pellet (1g) by a ratio thereof to prepare suspension of erythrocytes. The thusprepared E. coli suspension and erythrocyte suspension were mixed inequal quantities. The mixture (90 μl) was added to the 96-wellmicroplate (Corning Inc., U.S.A.), to which the sample (5 μl) and M9medium containing casamino acid (10 μl) were previously added. The platewas centrifuged at 1500 rpm for 10 minutes, and the hemolytic reactionwas initiated at 37° C. for 90-150 minutes. After the reaction, cooledPBS(−) (150 μl) (sodium chloride 0.8% (Kanto Chemical Inc., Japan),sodium dihydrogen phosphate 0.115% (Kanto Chemical Inc., Japan),potassium dihydrogen phosphate 0.02% (Wako Pure Chemical IndustriesLtd., Japan) and potassium chloride 0.02% (Kanto Chemical Inc., Japan))was added to prepare the suspension, and was centrifuged at 1500 rpm for10 minutes. Supernatant (100 μl) obtained by the centrifugation wastransferred into another 96-well microplate (Corning Inc., U.S.A.).Eluted hemoglobin was measured at 550 nm by using automatic microplatereader (Bio-Instruments Inc., U.S.A.). The hemolytic inhibitory activityis calculated by the following equation.Inhibition rate (%)=100−[(A−C)/(B−C).times.100]wherein

-   A: data at 550 nm when added the sample-   B: data at 550 nm of the mixture of erythrocyte and    enteropathogenic E. coli CesT defective strain alone-   C: data at 550 nm of erythrocytes alone

Results are as follows.

The drug concentration causing 50 % hemolytic inhibitory activity (IC₅₀)for K01-0509-A1 substance is 4.2 μg/ml and IC₅₀ for K01-0509-A2substance is 3.2 μg/ml. Both substances were demonstrated to inhibittype III secretion system.

(2) Assays of Antibiotic Activities for Enteropathogenic E. coli andVarious Test Microorganisms by Paper Disc Method

Test microorganisms used were enteropathogenic E. coli E2348/69 (wildstrain), Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC 9341, E.coli NIHJ, and Xanthomonas campestris pv. oryzae KB88. Medium used wasnutrient agar medium (peptone 0.5% (Kyokuto Pharmaceutical IndustrialCo., Ltd., Japan), meat extract 0.5% (Kyokuto Pharmaceutical IndustrialCo., Ltd., Japan) and agar 0.8% (Shimizu Shokuhin K.K., Japan), adjustedat pH 7.0). Activity was evaluated by paper disc method (diameter 6 mm:Advantech Co. Ltd.), and inhibitory zone was measured after 24 hourscultivation.

As a result, no inhibition zone was shown in all test microorganisms forK01-0509-A1 at 10 μg/disc and K01-0509-A2 at 10 μg/disc.

As described in detail, K01-0509 substance of the present invention canbe expected as novel anti-infectious disease drug, which inhibitsselectively type III secretion system without exhibiting antibacterialactivity.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows ultraviolet absorption spectrum of K01-0509-A1 substance(in water).

FIG. 2 shows infrared absorption spectrum of K01-0509-A1 substance (KBrtablet).

FIG. 3 shows proton nuclear magnetic resonance spectrum of K01-0509-A1substance (in deuterium oxide).

FIG. 4 shows carbon nuclear magnetic resonance spectrum of K01-0509-A1substance (in deuterium oxide).

FIG. 5 shows ultraviolet absorption spectrum of K01-0509-A2 substance(in water).

FIG. 6 shows infrared absorption spectrum of K01-0509-A2 substance (KBrtablet).

FIG. 7 shows proton nuclear magnetic resonance spectrum of K01-0509-A2substance (in deuterium oxide).

FIG. 8 shows carbon nuclear magnetic resonance spectrum of K01-0509-A2substance (in deuterium oxide).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained by illustrating example, but thepresent invention is not limited within the example.

EXAMPLE

A loopful of the strain K01-0509 cultured by an agar slant medium(starch 1.0% (Wako Pure Chemical Industries Ltd., Japan), N-Z amine 0.3%(Wako Pure Chemical Industries Ltd., Japan), meat extract 0.1% (KyokutoPharmaceutical Industrial Co., Ltd., Japan), CaCO₃ 0.3% (Kanto ChemicalInc., Japan) and agar 1.2% (Shimizu Shokuhin K.K., Japan), adjusted atpH 7.0) was inoculated into a medium (100 ml) (starch 2.4% (Wako PureChemical Industries Ltd., Japan), glucose 0.1% (Wako Pure ChemicalIndustries Ltd., Japan), peptone 0.3% (Kyokuto Pharmaceutical IndustrialCo., Ltd., Japan), yeast extract 0.5% (Oriental Yeast Co., Ltd., Japan)and CaCO₃ (Kanto Chemical Inc., Japan), adjusted at pH 7.0) in a 500-mlErlenmeyer flask and cultured at 27° C. for 3 days by using rotaryshaker (210 rpm) to obtain seed culture liquid. The seed culture (200ml) was inoculated into a production medium (20 liters) (starch 2.4%(Wako Pure Chemical Industries Ltd., Japan), glucose 0.1% (Wako PureChemical Industries Ltd., Japan), peptone 0.3% (Kyokuto PharmaceuticalIndustrial Co., Ltd., Japan), meat extract 0.3% (Kyokuto PharmaceuticalIndustrial Co., Ltd., Japan), yeast extract 0.5% (Oriental Yeast Co.,Ltd., Japan), calcium carbonate 0.4% (Kanto Chemical Inc., Japan), ironsulfate 7 hydrate 5.0×10⁻⁴% (Kanto Chemical Inc., Japan), magnesiumchloride 4 hydrate 5.0×10⁻⁴% (Wako Pure Chemical Industries Ltd.,Japan), copper sulfate 5 hydrate 5.0×10⁻⁴% (Kanto Chemical Inc., Japan)and cobalt chloride 6 hydrate 5.0×10⁻⁴% (Wako Pure Chemical IndustriesLtd., Japan)) in a. 30-L jar-fermenter, and cultured at 37° C. for 4days.

The medium cultured for 4 days (a total of 54 liters) was centrifuged byusing Sharpless centrifuge to separate into the supernatant andmicrobial cells. The supernatant was charged on a column of activecarbon (φ75×150 mm, Wako Pure Chemical Industries Ltd., Japan), washedwith water (1.5 liter), eluted the active principle with 20 and 40%acetone (each 1.5 liter), concentrated in vacuo and lyophilized. Theobtained crude substance (21.5 g) dissolved in a small amount of waterwas charged on a column of Amberlite IRC-50 (H+) (φ46×110 mm, OrganoCorp., Japan), washed with water (300 ml). The active principle waseluted with 1N HCl (600 ml), neutralized, desalted by means ofelectrodialysis, concentrated in vacuo and lyophilized to obtain crudeextract (823 mg).

The crude extract (200 mg) was dissolved in a small amount of water, andcharged on an ODS column (φ10×30 mm, Senshu Scientific Co., Ltd., Japan)equilibrated with 0.1% aqueous trifluoroacetic acid solution, washedwith 0.1% aqueous trifluoroacetic acid solution (10 ml), eluted theactive principle with 20% methanol in 0.1% aqueous trifluoroacetic acidsolution (5 ml), concentrated in vacuo and lyophilized. The remainedcrude substance (623 mg) was treated by the same manner to obtain activesubstance (103 mg). The active substance (103 mg) was dissolved in asmall amount of water, and was purified by using the preparative HPLC(column: Develosil C30-UG-5, φ20×250 mm, Nomura Chemical Co., Ltd.,Japan). The UV absorption at 210 nm was monitored in the isocraticmobile phase of 8% methanol in 0.1% aqueous trifluoroacetic acidsolution at flow rate of 5 ml/min. A peak showing activity at theretention time of 88 min. was observed and collected. The collectedsolution was concentrated in vacuo and lyophilized.

The obtained active material (8.3 mg) was dissolved in a small amount ofwater and purified by using the preparative HPLC (column: DevelosilC30-UG-5, φ20×250 mm, Nomura Chemical Co., Ltd., Japan). The UVabsorption at 210 nm was monitored in the isocratic mobile phase of 3%acetonitrile in 0.05% aqueous phosphoric acid solution at flow rate of 5ml/min. Peaks showing activity at the retention times of 27 min. and 30min. were observed and collected. The collected solution wasconcentrated in vacuo, and charged on the ODS column (φ5×10 mm, SenshuScientific Co., Ltd., Japan) equilibrated with 0.1% aqueoustrifluoroacetic acid solution, washed with 0.1% aqueous trifluoroaceticacid solution (5 ml), eluted the active substance with 100% methanolwith 0.1% (final concentration) trifluoroacetic acid (5 ml) concentratedin vacuo and lyophilized to obtain white powdery K01-0509-A1 substance(1.1 mg) and K01-0509-A2 substance (1.5 mg).

INDUSTRIAL APPLICABILITY

As explained hereinabove, the microorganism represented by the strainK01-0509, having ability to produce K01-0509-A1 substance andK01-0509-A2 substance, belonging to genus Streptomyces is cultured in amedium, and K01-0509-A1 substance and K01-0509-A2 substance havinginhibitory activity against type III secretion system are isolated.These substances can be expected as the selective and effectivepharmaceuticals for treatment or prevention of infection withenteropathogenic gram negative bacteria having type III secretionsystem.

1. K01-0509-A1 substance represented by the formula [I];


2. K01-0509-A2 substance represented by the formula [II];

which is a stereo isomer of K01-0509-A1 substance.
 3. A composition ofK01-0509 substance consisting of specifically K01-0509-A1 substancerepresented by the formula [I];

and/or specifically K01-0509-A2 substance represented by the formula[II];

which is a stereo isomer of K01-0509-A1 substance.
 4. A process forproduction of K01-0509-A1 substance, which is described in claim 1,comprising culturing a microorganism belonging to genus Streptomyces andhaving ability to produce K01-0509-A1 substance in a medium,accumulating K01-0509-A1 substance in the cultured medium and isolatingK01-0509-A1 substance from the cultured mass.
 5. A process forproduction of K01-0509-A2 substance, which is described in claim 2,comprising culturing a microorganism belonging to genus Streptomyces andhaving ability to produce K01-0509-A2 substance in a medium,accumulating K01-0509-A2 substance in the cultured medium and isolatingK01-0509-A2 substance from the cultured mass.
 6. A process forproduction of the composition, which is described in claim 3, comprisingculturing a microorganism belonging to genus Streptomyces and havingability to produce K01-0509-A1 substance and/or K01-0509-A2 substance ina medium, accumulating K01-0509-A1 substance and/or K01-0509-A2substance, in the cultured medium and isolating K01-0509-A1 substanceand/or K01-0509-A2 substance from the cultured mass.
 7. The process forproduction of K01-0509-A1 substance according to claim 4 wherein themicroorganism belonging to genus Streptomyces and having ability toproduce K01-0509-A1 substance is Streptomyces sp. K01-0509 FERMBP-08504.
 8. The process for production of K01-0509-A2 substanceaccording to claim 5 wherein the microorganism belonging to genusStreptomyces and having ability to produce K01-0509-A2 substance isStreptomyces sp. K01-0509 FERM BP-08504.
 9. The process for productionof the composition consisting of and/or K01-0509-A2 substance accordingto claim 6 wherein the microorganism belonging to genus Streptomyces andhaving ability to produce K01-0509-A1 substance and/or K01-0509-A2substance is Streptomyces sp. K01-0509 FERM BP-08504.
 10. Amicroorganism Streptomyces sp. K01-0509 FERM BP-08504.
 11. Themicroorganism according to claim 10 wherein the microorganism is mutantof Streptomyces sp. K01-0509 FERM BP-08504.